Methylenetetracycline formate ester

ABSTRACT

1. 6-DEMETHYL-6:DEOXY-6:METHYLENE-5-FORMYLOXY-TETRACYCLINE.

United States Patent Oifice Patented Oct. 15, 1974 US. Cl. 260-490 1Claim ABSTRACT OF THE DISCLOSURE 6 Demethyl 6 deoxy 6methylene-5-acyloxytetracyclines having antibiotic activity againsttetracycline resistant strains. Also disclosed are the intermediatesllachloro 6 demethyl 6 deoxy 6 methylene 5 acyloxy-tetracyclines. Modesof preparation are given.

The present invention has as its object methylenetetracycline esters,their pharmaceutically acceptable non toxic acid addition salts and aprocess for the preparation thereof.

More particularly, the new tetracycline derivatives have the structure:

wherein R is a radical of an organic acid containing from 1 to 10 carbonatoms and X is selected from the group consisting of hydrogen, chlorideand bromine and they have interesting antibacterial activity. Thisantibiotic activity against tetracycline resistant strains or good oraladsorption, are characteristic of these compounds.

The process for the preparation of the new esters consists in treating11a-chloro-6-demethyl-6-deoxy-6-methylene-S-hydroxy-tetracycline III asthe free base or its salt prepared as described and claimed in US.application Serial No. 48,521, filed June 22, 1970, claiming priority ofItalian Patent Application No. 18792 A/69, filed June 27,1969 byapplicants, and now abandoned with an organic acid containing in itsmolecule from 1 to 10 carbon atoms in the presence of an agent selectedfrom the group consisting of methanesulfonic, ethanesulfonic andhydrofluoric acids at a temperature between 20 and 70 C., over a periodof from 2 to 20 hours.

The ester H thus obtained is then reduced to give the correspondingdechlorinated ester I. The reducing agent may consist of zinc in an acidmedium, sodium and potas- 6 sum hydrosulfites or hydrogen in thepresence of catalysts. The process of the invention may be representedas follows:

Meir

I I contr o l x n! a (III) Q N(CK X cs E Esteriiication 5 OH com:

l 3 tr l in. on

Reduction Alternatively, the intermediate compounds of Formula II may beprepared by reacting an lla-chloro-S-hydroxytetracycline-6,IZ-hemiketalof this formula:

(III!) wherein X is hydrogen, chlorine or bromine with a mixtureconsisting of an organic acid having from 1 to 10 carbon atoms and analkylsulfonic acid such as methanesulfonic acid or ethanesulfonic acid.The reaction is carried out at a temperature between 5 and 50 C., over aperiod from 5 to 25 hours. The lla-chloro-6-demethyl-6-deoxy-G-methylene-S-hydroxy-tetracycline-S-monoester is isolated in theform of the free base or one of its salts.

According to this alternative, the complete sequence for preparingcompound of Formula (I) is:

go y,-

wherein R and X are as defined above.

Obviously, the alternative sequence gives better yields since thepreparation of the intermediate compound III from III can be avoided.The compounds (I) of the invention are particularly interesting asanti-bacterial drugs. Many of them develop a very strong antibacterialactivity on tetracycline-resistant strains, while others show very goodoral adsorption and give blood levels, which are higher than that givenby metacycline or 6-demethyl-6- deoxy-6-methylene-5-hydroxy-tetracyclinewith which the compounds of the invention have a close structuralanalogy.

The activity against tetracycline resistant strains was tested in vitroin comparison with that shown by metacycline. Table I reports the valuesof the MIC or Minimal Inhibiting Concentration (in ,ugjml.) which represents the minimum quantity of the substance capable of inhibitingcompletely, in vitro, the development of the tested organism.

TABLE I MIC, gJml.

fi-demethyl-G 4 deoxy-6-methylene-fi-isobutyr- Strains oi clinicisolation Metacyiloxy- Staphylococcus aureul eline tetracycline Table IIreports the activity against tetracycline resistant Staphylococcusaureus ATCC 12715.

TABLE 11 Compound: MIC, gjml.

6 demethyl 6 deoxy-6-methylene-5-oxytetracycline (metacycline) 20 6demethyl 6 deoxy-6-methylene-S-acetyloxy tetracycline 5 6 demethyl 6deoxy-6-methylene-5-propionyloxy tetracycline 2.5 6 demethyl 6deoxy-G-methyIene-S-butyrriloxy tetracycline 2.5

4 TABLE II-Continued Compound: MIC, ug/ml.

6 demethyl 6 deoxy-6-methylene-5-isobutyrryloxy tetracycline 0.62

6 demethyl 6 deoxy-6-methylene-5-pivalyloxy tetracycline 0.62

6 demethyl 6 deoxy-6-methylene-5-diethylacetyloxy tetracycline 0.62

6 demethyl 6 deoxy-6-methylene-5-capryliloxy tetracycline 0.62

Table III reports some results of a comparison trial in viva betweenmetacycline and the first member of the compounds of the presentinvention, i.e. 6-demethyl-6- deoxy 6methylene-5-formyloxy-tetracycline. This test was carried out in themouse (12 animals per group) infected intraperitoneously withStaphylococcus aureus PV and the tested antibiotics were orallyadministered 4, 24, 48 and 72 hours after the infection. The symbol PDmeans protective dose 50.

TAB LE III Mortality, percentage Dosage, by the Compound rug/kg. 11thday PD .1 Control.

Metacycllne- 40 2O 10 6-demethyl-6-deoxy-6-methy1ene-5-formyloxytetracycline- 20 TABLE IV gJml. in serum after- Compound 2hours 4 hours 6 hours Metacycline. 0. 41 0. 46 0. 46fi-demethyl-fi-deoxy-fi-methylene-5- formyloxytetracycline 1. 49 1. 270. 89

The following Examples are given to illustrate the invention without,however, limiting it.

EXAMPLE 1 6-Demethy-6-deoxy-6-methy1ene-S-acetyloxy-tetracyoline Asolution of 10 g. of 11a chloro-6-demethyl-6-deoxy- 6-methylene 5hydroxy-tetracycline p-toluenesulfonate (tosylate) in a mixture of 50cc. of glacial acetic acid and 50 cc. of 99100% methanesulfonic acid wasstirred at 45 50 C. for 4 hours, then poured into 1500 cc. of ether. Theoily, dark brown precipitate was filtered on an infusorial earth cake,then dissolved in methanol and decolored with charcoal. The methanolsolution was concentrated under reduced pressure to a small volume anddiluted with isopropanol. By further dilution with ether, the productseparated. This was filtered, washed several times with anhydrous etherand dried in vacuo over phosphoric anhydn'de. Thus, 8 g. of11a-chloro-6-demethyl-6- deoxy-6-methylene 5 acetyloxy-tetracyclinemesylate were obtained [R,=0.70 on Whatman paper no. 1 butfered at pH4.6 with McElvain butter and using as dilueat, the upper layer of thesolvent mixture: methylisobntyl-ketone-ethyl acetate-n. butanol-water(80:80:35: 88)]. This product may be used as such for the subse- QECOO62.0 S N(CH3)2 62.4 S =CH2 55.48 s

66.00 Aromatic protons 66.9-7.8 m.

The 5-sulfosalicylate has maxima in the LR. spectrum at 3340, 1765,1740, 1675, 1640, 1280, 1220, 1160 and 1050 cmr By N.M.R. (DMSO) itshows the following characteristic signals:

CHaCOO..-

mmmm

=CH1 Aromatic protons- 66:8-82 m.

Found percent: C, 50.10; H, 4.10; N, 4.19; O, 32.37; Cl, 4.88; S, 4.41.Calculated for C H ClN O -C H O S: C, 50.51; H, 3.97; N, 3.80; 0, 32.56;Cl, 4.81; S, 4.35.

1 g. of Ila-chloro 6 demethyl 6 deoxy-6-methylene 5acetyloxy-tetracycline free base (or its salt) was suspended in cc. ofmethanol-water (1:1) and 0.60 g. of sodium hydrosulfite were addedlittle by little. A solution was first obtained, which then becameturbid. After minutes, the solution was adjusted to pH 4 with N sodiumhydroxide, diluted with water and extracted with ethyl acetate. Theorganic extracts were washed with water, dried over sodium sulfate andevaporated under reduced pressure. Crystallization from ether-petroleumether yielded 0.69 g. of 6-dcmethyl 6 deoxy-6-methylene-5-acetyloxy-tetracycline, R =0.88.

The U.V. spectrum shows a maximum at 239 m;:., an inflexion at about 270my and another maximum at 343 my. in MeOH-HCl 0.01N (1:9); a maximum at234 mu, an inflexion at about 278 mg and another maximum at 382 m in0.01N NaOH. The N.M.R. spectrum (DMSO) shows the followingcharacteristic maxima:

CHfiCOO 62.1 s. N(CHa)z 62.45 s. =CH2 85.52 Broad. Aromatic protons66.87.7 m.

The compound is characterized as hydrochloride.

Analysis.-Calcd. for C H N O -HCl: C, 55.33; H, 4.85; N, 5.38; Cl, 6.8.Found (percent): C, 55.26; H, 5.21; N, 4.97; CI, 7.03.

EXAMPLE 2 6-Demethyl-6-deoxy-6-methylene-S-acetyloxytetracycline 18.4 g.of hydrofluoric acid were bubbled in 16 cc. of glacial acetic acid, then2 g. of 11a-chloro-6-demethyl- 6-deoxy 6 methylene 5hydroxy-tetracycline tosylate were added. The resulting solution wasallowed to stand for 18 hours at room temperature. It was then pouredinto a saturated aqueous solution containing 57 g. of calcium chloride.The precipitate was filtered through an infusorial earth cake and theaqueous solution was extracted three times with butanol. The butanolextracts, washed with a little water, were concentrated in vacuo to asmall volume. Dilution with petroleum ether yielded 1.4 g. of 11achloro6 demethyl 6 deoxy 6 methylene 5 acetyloxy-tetracycline tosylate which,after reduction as described in Example 1, gave 6-demethy1 6 deoxy-6-methylene-S-acetyloxy-tetracycline.

6 EXAMPLE 3 1 1a-Chloro-6-demethyl-6-deoxy-6-methylene-5-acetyloxy-tetracycline 50 g. of 11a chloro 5 hydroxy-tetracycline-6,12-hemiketal were suspended in 250 cc. of glacial acetic acid, then 250 cc.of methanesulfonic acid were added. The resulting solution was allowedto stand for 16 hours at room temperature. It was then poured into 10liters of ether. The semi-oily dark brown precipitate was filteredthrough an infusorial earth cake, then dissolved in butanol anddecolored with charcoal. The resulting clear light yellow solution wasconcentrated under reduced pressure and diluted with diethyl ether.After drying, 50- 55 g. of the crude mesylate of Ila-chloro 6demethyl-6- deoxy 6 methylene 5 acetyloxy-tetracycline were obtained: R=0.70 in chromatography on Whatman paper No. 1, buffered at pH 4.6 withMcElvain buffer and using as diluent the upper layer of the solventmixture: methylisobutylketone-ethyl acetate-n. butanol-water(:80:35288). (Farmaco Ed. PL, 17, 728 (1962)). To detect spots by theWood lamp, the dsh was previously sprayed with an aqueous solution of15% Na S O The free base, obtained from the mesylate by conventionaltechniques known in tctracyclines chemistry, shows adsorption maxima atthe U.V. at 238, 282, and 382 m,u. in CH OH:HCl 0.01N (1:9) and at 235,280 and 393 my in NaOH 0.01N The IR. spectrum (KBr) shows an absorptionmaximum between 3410 and 3458 cm.- and other maxima at 1755, 1650, 1612,1535, 1230 cmf The N.M.R. spectrum (DMSO) shows the followingcharacteristic signals:

13000 52.0 s N(CH3)2 S :(JH2 65-48 g The 5-sulfosalicylate hasabsorption maxima at the IR. spectrum at 3340, 1765, 1740, 1675, 1640,1280, 1220, 1160 and 1050 cmr The NMR (DMSO) spectrum shows thefollowing characteristic signals:

E3000 52.1 s N(C a)z 22% g =CH2 5610 s Aromatic protons 66.8-82 m.

EXAMPLE 4 6-Dernethyl-6-deoxy-6-methylene-5-propionyloxytetracycline2113-0112-000 61.05 t J=6.5 cps. N(OH3)1 63.2 s 5 s "{aao s Aromaticprotons 56.8-55.2 111.

Ila-chloro 6 demethyl 6 deoxy 6 methylene-5 propionyloxy-tetracyclinemesylate was then reduced as previously described and 6-demethyl 6 deoxy6 methylene 5 propionyloxy-tetracycline hydrochloride was obtained, R=0.92.

The U.V. spectrum shows a maximum at 240 me, an inflexion at 270 m andanother maximum at 342 m, in 0.01N MeOH-HCl (1:9).

7 The N.M.R. spectrum (DMSO) hydrochloride shows the followingcharacteristic maxima:

CHaCHzCOO 61.1 t .T=6. 5 cps.

N(CHQ)2 62.9 s =CH2 65.4 Broad--. Aromatic protons 56.8-7.7 m.

At the analysis.--found (percent): C, 56.91; H, 5.23; N, 5.36. Calcd.for C H N O -HCl: C, 56.12; H, 5.10; N, 5.24.

EXAMPLE 5 1 1a-Chloro-6-demethyl-6-deoxy-6-methylene-5-propionyloxy-tetracycline Operating as described in Example 3, butemploying propionic acid instead of acetic acid,11a-chloro-6-demethyl-6-deoxy-6-methylene-5-propiony1oxy tetracyclinemesylate was obtained (R =0.80) and subsequently the free base.

EXAMPLE 6 6-Demethyl-6-deoxy-6-methylene-5-butyryloxytetracyclineOperating as in Example 1 and employing butyric acid instead of aceticacid, 1la-chloro-6-demethyl-6-deoxy-6-methylene-S-butyryloxy-tetracycline mesylate is obtained; R;=0.9. Theproduct was purified as the 5-sulfosalicylate.

Analysis.Found (percent): C, 53.74; H, 4.60; N, 4.08; CI, 5.49; S, 2.76.Calcd. for

(percent): C, 54.00; H, 4.62; N, 4.27; CI, 5.40; S, 2.44. The N.M.R.spectrum (DMSO) shows the following characteristic maxima:

Aromatic protons c6.8-8.2m:

11a-chloro-6-demethyl-6-deoxy-6-methylene 5 butyryloxy-tetracyclinemesylate was then reduced as previously described and6-demethyl-6-deoxy-6-methylene-5- butyryloxy-tetracycline was obtained,R =0.94. The U.V. spectrum shows a maximum at 240 m an inflexion at 270my and another maximum at 344 my in 0.01N MeOH-HCI (1:9). The N.M.R.spectrum (DMSO) for the free base shows the following characteristicmaxima:

=H, 65.5 Aromatic protons a6.8-7.7m.

Analysis.Calcd. for C d-1 N 0 (percent): C, 60.92; H, 5.52; N, 5.47; O,28.09. Found (percent): C, 60.62; H, 5.52; N, 4.89; O, 27.71.

EXAMPLE 7 1 la-Chloro-6-demethyl-6-deoxy-6-methylene-5-butyryloxy-tetracycline Operating as described in Example 3, butemploying butyric acid instead of acetic acid, lla-chloro-6-demethyl-6-deoxy-6-methylene-5-butyryloxy-tetracyc1ine mesylate was obtained,(Rg=0.90) and subsequently the free base.

EXAMPLE 8 6-Demethyl-6-deoxy-6-methylene-5-isobutyryloxytetracyclineOperating as in Example 1 and employing isobutyric acid instead ofacetic acid there was obtained Ila-chloro-6-demethyl-6-deoxy-G-methylene isobutyryloxy-tetracycline mesylate whichwas then reduced to 6-demethyl- 6-deoxy-6-methylene 5 isobutyryloxytetracycline,

8 R =0.93. The U.V. spectrum in CH OH-HCl 0.01N shows maxima at 240 m275 inflexion, 345 mg. The I.R. spectrum shows maxima at 1740, 1230 cm."The N.M.R. spectrufrn (DMSO) shows the following characteristic signals:

(CH3)2CH v1.11 (1 N(CH3)1 62.9 S =CH2 474 Aromatic portons 0'6.87.7 m.

At the analysis.-Calcd. for C H N o -HCl (percent): 0, 56.87; H, 5.33;N, 5.10; 0, 26.23; 01, 6.46. Found (percent): C, 56.66; H, 5.43; N,5.09; O, 26.29; 01, 6.50.

EXAMPLE 9 1 1a-Chloro-6-demethyl-6-deoxy-6-methylene-5-isobutyryloxy-tetracycline Operating as in Example 3, but employingisobutyric acid instead of acetic acid, 11a-chloro-6-demethyl-6-deoxy-6-methylene-5-isobutyryloxy-tetracycline mesylate was obtained aswas subsequently the free base.

EXAMPLE 10 6-Demethyl-6-deoxy-6-methylene-5-pivalyloxytetracyclineOperating as in Example 1 and employing pivalic acid instead of aceticacid, l1a-chloro-6-demethyl6-deoxy-6-methylene-S-pivalyloxy-tetracycline mesylate was obtained which, onreduction, gave 6-demethyl-6-deoxy-6-methy1-ene-5-piva1yloxy-tetracycline. The U.V. spectrum in 0.01N CH OH-HC1shows maxima at 239, 273 inflexion and 344 me. The LR. spectrum (KBr)shows maxima at 1740 and 1230 cmr' The N.M.R. spectrum (CDCl shows thefollowing characteristic maxima:

C(CH3)3 51.21 S N( a)i i-3 a J 2 a cps. 55.35 d J'=2 cps. Aromaticprotons 67.76.7

EXAMPLE 11 1 la-Chloro-6-demethy1-6-deoxy-6-methy1ene-5-pivalyloxy-tetracycline Operating as described in Example 3, butemploying pivalic acid instead of acetic acid, 11a-chloro-6-demethyl-6-deoxy-6methylene 5 pivalyloxy-tetracycline mesylate and subsequentlythe free base were obtained.

EXAMPLE 12 6-Demethyl-6-deoxy-6-methylene-5-caprylyloxytetracycline CHCHzCHz-CH1CHCHCHz--- 513 Broad. N(C'H'1)a 62.5 S. =CH 65.5 Broad.

EXAMPLE 13 1 1a-Chloro-6-demethyl-6-deoxy-6-methylene-5-caprylyloxy-tetracycline Operating as in Example 3, but employingcaprylic acid instead of acetic acid, lla chloro-6-demethyl-6- deoxy 6methylene--capryly1oxy-tetracycline mesylate and subsequently the freebase were obtained.

EXAMPLE 14 6-Demethyl-6-deoxy-6-methylene-5-diethylacetyloxytetracyclineEXAMPLE 15 1 1a-Chloro-6-demethyl-6-deoxy-6-methylene-5-diethylacetyloxy-tetracycline Operating as described in Example 3, butemploying diethylacetic acid instead of acetic acid, 11a-chloro-6-demethyl 6 deoxy-6-methylene-5-diethylacetyloxy-tetrau cycline mesylateand subsequently the free base were obtained.

EXAMPLE 16 6-Demethy1-6-deoxy-6-methylene-5-formyloxytetracycline 20 g.of 11a-chloro-6-demethyl-6-deoxy-6-methyleneoxytetracyclinepara-toluenesulfonate and 100 cc. of 99% formic acid were poured into apolyethylene lined vessel. Then about 100 cc. of anhydrous hydrofluoricacid were added and the mixture was allowed to stand overnight at roomtemperature. A nitrogen flow was bubbled through the obtained product soas to remove the hydrofluoric acid. The residue was poured into about 2liters of ethyl ether. It was stirred for 10 minutes and then filtered.The filtered cake was washed with ethyl ether and dried under vacuum.16.9 g. of product are obtained as the paratoluenesulfonate.

16.9 g. of 11a-chloro-6-demethyl-6-deoxy-6-methylene- 5-formyloxytetracycline paratoluenesulfonate were dissolved in a glass containing300 cc. of a methanol/water solution. 8.5 g. of sodium hydrosulfite wereadded and the mixture was allowed to stand at room temperature forminutes. The pH of the mixture was adjusted to 4, the methanol wasevaporated oh and the residue was extracted 3-4 times with ethylacetate. The extract was then washed with water and dried over anhydroussodium sulfate. The solution was brought to a small volume, and byadding petroleum ether, a substance precipitated. This substance wasfiltered, Washed with petroleum ether and dried under vacuum. 9.6 g. ofproduct were obtained as the free base, of6-demethyl-6-deoxy-6-methylene tetracycline, which was about S-10%impure. The crude product was dissolved in about 1 00 cc. of the upperlayer of the following solvent mixture: methylisobutylketone-ethylacetate-n. butanol-0.1N citric acid-0.1N dibasic sodium phosphate,48:48:21:55:55.

The thus obtained solution was shaken 2-3 times with the lower layer ofsaid solvent mixture. It was then taken up with ethyl acetate, washed2-3 times with water, and dried over anhydrous sodium sulfate. Afterdecoloration with charcoal, the product was brought to a small volumeand precipitated with petroleum ether. 7.3 g. of pure product wereobtained (Yield 58% The U.V. spectrum [solution in CH OH/HCl 0.01N(1:9)] shows the following maxima:

)\m n 240 mp. (e= 20, 173.) 273 mp. (e= 17, 079.) 343 mp (e=11,881.)

The LR. spectrum (in KBr) shows the following bands at 1730 and 1170cm.- characteristic of esters.

The N.M.R. in CD01 shows the following characteristic maxlma:

-C=O (formlate) 68.14

N(CH3): 62.49 s =CH2 55.44 2d Aromatic protons- 6680-710 C5-H 65.93 2dThe invention also includes the hitherto unknown compounds II which areintermediates in the preparation of compounds of the formula I. The termdeoxy" as used herein is also often called desoxy.

What is claimed is:

1. 6 demethyl-G-deoxy-6-methylene-5-formy1oxy-tetracycline.

References Cited UNITED STATES PATENTS 3,579,564 5/1971 Blackwood et al260473 3,047,617 7/1962 Blackwood et al 260490 2,812,349 11/1957 Gordon260490 3,069,467 12/1962 Beereboom et al 260559 3,272,817 9/ 1966 Gordonet a1 260490 OTHER REFERENCES Merck Index, 1968, 668-669. Chem.Abstracts, 5 8: 4079d. Chem. Abstracts, 58: 12487a. Chem. Abstracts, 60:482f.

VIVIAN GARNER, Primary Examiner US. Cl. X.R.

260346.2M, 410, 559AT; 424-227 qggg UN TED STATES PATENT OFFICECERTIFICATE OF CORRECTION Patent No. 3,842,1 3 Dated October 15, 1974Inventor(s) Luigi BERNARDI et al It is certified-that error appears inthe above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 1, line 42: "chloride" should read chlorine Column 2, line 39:"this" should read the Column 3, line 2: "compound" should readcompounds line 32: After last formula should read I Table I, column 3,in the heading: "5isobutyriloxy-" should read 5-isobutyrriloxy- Column4, line 1?: "administered 4, 24, should read administered; at 4, 24,

Column 6, line 2 2: "dsh" should read dish line 28 "O.OlN should read0.01N. line 37: "5 6.97- 7.8m. should read 6.9-7.8m.

Column 8, line 10: "Calcd. for" should read Calcd. Z for lines lO-ll:delete "(percent)".

Column 10, line 20: "273 m u." should read 274 r.

Signed and sealed this 11th day of March 1975.

(SEAL) Attest:

c. MARSHALL DANN RUTH C MASON Commissioner of Patents Attest ng Offlcerand Trademarks

1. 6-DEMETHYL-6:DEOXY-6:METHYLENE-5-FORMYLOXY-TETRACYCLINE.